high altitude hypoxia as a factor that promotes tibial growth ......citation: huang s, zhang l,...

RESEARCH ARTICLE

High altitude hypoxia as a factor that

promotes tibial growth plate development in

broiler chickens

Shucheng Huang1, Lihong Zhang1, Mujeeb Ur Rehman1, Muhammad Kashif Iqbal1,

Yanfang Lan1, Khalid Mehmood1, Hui Zhang1, Gang Qiu1,2, Fazul Nabi1,3, Wangyuan Yao1,

Meng Wang1, Jiakui Li1,2*

1 College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, People’s Republic of China,

2 Laboratory of Detection and Monitoring of Highland Animal Disease, Tibet Agriculture and Animal

Husbandry College, Linzhi Tibet, People’s Republic of China, 3 Faculty of Veterinary & Animal Sciences,

Lasbela University of Agriculture, Water and Marine Sciences Uthal, Balochistan, Pakistan

* [email protected]

Abstract

Tibial dyschondroplasia (TD) is one of the most common problems in the poultry industry

and leads to lameness by affecting the proximal growth plate of the tibia. However, due to

the unique environmental and geographical conditions of Tibet, no case of TD has been

reported in Tibetan chickens (TBCs). The present study was designed to investigate the

effect of high altitude hypoxia on blood parameters and tibial growth plate development in

chickens using the complete blood count, morphology, and histological examination. The

results of this study showed an undesirable impact on the overall performance, body weight,

and mortality of Arbor Acres chickens (AACs) exposed to a high altitude hypoxic environ-

ment. However, AACs raised under hypoxic conditions showed an elevated number of red

blood cells (RBCs) and an increase in hemoglobin and hematocrit values on day 14 com-

pared to the hypobaric normoxia group. Notably, the morphology and histology analyses

showed that the size of tibial growth plates in AACs was enlarged and that the blood vessel

density was also higher after exposure to the hypoxic environment for 14 days, while no

such change was observed in TBCs. Altogether, our results revealed that the hypoxic envi-

ronment has a potentially new role in increasing the blood vessel density of proximal tibial

growth plates to strengthen and enhance the size of the growth plates, which may provide

new insights for the therapeutic manipulation of hypoxia in poultry TD.

Introduction

The Tibetan plateau is a high altitude geographical region of China with an average elevation of

more than 4,000 meters. This region is generally known for its extreme environmental condi-

tions, including low oxygen content, low barometric pressure, and great temperature fluctuations

on a daily basis. These conditions impose severe physiological challenges on endothermic animals

[1,2]. The typical stress at high altitudes is hypoxia, which is caused by the fall in barometric

PLOS ONE | DOI:10.1371/journal.pone.0173698 March 10, 2017 1 / 14

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OPENACCESS

Citation: Huang S, Zhang L, Rehman MU, Iqbal

MK, Lan Y, Mehmood K, et al. (2017) High altitude

hypoxia as a factor that promotes tibial growth

plate development in broiler chickens. PLoS ONE

12(3): e0173698. doi:10.1371/journal.

pone.0173698

Editor: Bi-Song Yue, Sichuan University, CHINA

Received: November 4, 2016

Accepted: February 25, 2017

Published: March 10, 2017

Copyright: © 2017 Huang et al. This is an open

access article distributed under the terms of the

Creative Commons Attribution License, which

permits unrestricted use, distribution, and

reproduction in any medium, provided the original

author and source are credited.

Data Availability Statement: All relevent data are

within the paper and its supporting informtion files.

Funding: The study was supported by The National

Natural Science Foundation of China (No.

31460682). Funds received by Jiakui Li.

Competing interests: The authors have declared

that no competing interests exist.

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http://creativecommons.org/licenses/by/4.0/

pressure with increasing altitude and consequently lower oxygen content in the inspired air com-

pared to sea level [1]. Furthermore, due to the slow growth rate of Tibetan chickens (TBCs), it

has been a common practice for most poultry farmers in Tibet to raise commercial broiler chick-

ens (such as Arbor Acres chickens) because of their fast growth rate. However, these chickens are

not well adapted to the hypoxic environment, which is the main ecological factor with a negative

impact on the animal’s health and a threat to their survival at high altitudes [3].

Hypoxia refers to low partial pressure of oxygen (O2) in the inspired air and threatens the

survival, development, and reproduction of both humans and animals [3–10] because of its

physiological challenges on the body. Jia et al. [10] have revealed the unique physiological

responses and adaptation mechanism of animals in response to high altitude hypoxia. Chick-

ens provide a proper model to study physiological adaptations under hypoxic stress condi-

tions, and TBCs are a unique aboriginal breed that has undergone selection for such trials to

inhabit the high altitude Tibetan plateau. TBCs are one of the native poultry breeds that have

been found on the Tibetan plateau (2,600 m~4,500 m above sea level) for approximately 1000

years. Therefore, this breed has the ability to adapt to rigorous environmental conditions such

as low air pressure and partial pressure of oxygen [11–13].

In comparison to the chicken breeds at lower altitudes, TBCs have the adaptability to sur-

mount the extremely harsh environments due to their elevated number of red blood cells and

blood hemoglobin level [11]. Most importantly, this unique breed has never been reported to

have any leg disorders, especially Tibial dyschondroplasia (TD). There is a high incidence rate

of TD in meat-type and fast-growing poultry, especially turkeys, with up to 80% developing

TD at the age of 12 weeks [14]. Numerous studies have reported that TD is a bone abnormal-

ity. The lesions of TD are characterized by the presence of an irregular, white, opaque, unmi-

neralized and unvascularized mass of cartilage that is attributable to cell death, no blood

supply and degenerative changes in the proximal end of the tibia and that is the leading cause

of osteomyelitis, osteochondrosis, and lameness in poultry [14–19]. Altogether, these factors

lead to significant economic losses to the poultry industry and compromise poultry welfare. In

addition, it has been reported that normal avian growth plates consist of long columns of

chondrocytes that are well vascularized with more cellular zones compared to mammalian

growth plates [19–21]. Moreover, the growth plate regulates bone ossification and elongation

by maintaining the balance between chondrocyte proliferation and differentiation [22,23].

However, the etiology of TD linked to the development of the growth plate is still unknown.

Rath et al. [16] proposed that the possible pathogenesis of TD is linked to abnormal cell death

(apoptosis) in the growth plates, and these dead chondrocytes cannot be removed promptly

due to sparse vascularity of the growth plate.

Numerous studies have highlighted the role of hypoxia in initiating the expression of

hypoxia-induced factor-1 (HIF-1α) and further inducing the expression of target genes

such as vascular endothelial growth factor (VEGF) and its receptors, which promote various

systemic physiological changes including angiogenesis and vascular development [24–29].

Angiogenesis plays a crucial role in the homeostatic mechanisms associated with the vascu-

lar oxygen supply in hypoxia. However, the mechanism of hypoxia-induced tibial growth

plate development and function remains unclear. Therefore, this study was designed to

understand the physiological mechanism of hypoxia in the development of the growth plate

and to understand the possible association of angiogenesis and vascular development mech-

anisms with high altitude hypoxia during the early stages of broiler growth in AACs and

TBCs, which may provide new insights for the therapeutic manipulation of hypoxia to pre-

vent lameness in these birds.

Hypoxia promotes the development of tibial growth plates


Materials and methods

Ethics statement

All the experiments were approved and reviewed by the Animal Welfare and Ethics Committee

of the Huazhong Agricultural University Wuhan, China (approval permit number: 31272517).

The animal experiments and procedures were performed in strict accordance with the relevant

guidelines of PSCH (No. 5 Proclamation of the Standing Committee of Hubei People’s Congress,

P.R. China). Notably, none of the chickens exhibited signs of illness or distress prior to their

death. However, to minimize suffering, the chickens were euthanized using pelltobarbitalum

injections with standard protocols if they exhibited specific signs of illness during the experiment.

Chicken husbandry

One-day-old healthy AACs (n = 120) were purchased from a commercial hatchery of

Chengdu, China (average altitude, 500 m), and transported to a laboratory of the Tibet Agri-

cultural and Animal Husbandry College (average altitude, 2,986 m above sea level) on the

same day. Simultaneously, one-day-old healthy TBCs (n = 120) were also purchased from a

commercial hatchery at Lhasa (average altitude, 3,651 m) and transported to the same labora-

tory. All the chicks were randomly allocated into two groups by birth weight, namely, the

hypobaric normoxia group and the hypoxia group (approximately 21% oxygen content and

natural oxygen content, respectively; n = 60/group, 4 cages per treatment and 15 chicks per

cage). The oxygen content of the hypobaric normoxia group was maintained with an oxygena-

tor (Yuwell, Suzhou, China). Moreover, the oxygen content of the normoxia and hypoxia

groups was monitored with a gas detector (CY-7B, Oxygen analysis instrument factory, Jiande,

China) throughout the experiment.

All the AACs (40.5±1.02 g) and TBCs (31.2±2.03 g) had similar initial weights or birth

weights (Fig 1A), and the nutrient contents of the diets (12.6 MJ metabolizable energy/kg of

diet, 220 g/kg crude proteins) were maintained as suggested by the National Research Council

(NRC, 1994). The nutrient composition of the broiler diet is shown in S1 Table. The chicks

were raised in two-layer metal cages (size, 80 cm×60 cm×50 cm) for 14 days. Four times a day

(every 6 h), their diet, drinking water, and overall performance were monitored; lack of clus-

tering and no difficulty in breathing were considered normal performance for the chickens.

The brooding temperature was maintained between 33˚C and 35˚C during the first week and

gradually decreased up to 29˚C by the end of second week. The daily light/dark cycle was fixed

at 23 h light and 1 h dark during the whole experiment. In addition, feed and water were pro-

vided ad libitum.

Production performance analysis

The chicks were group-weighed on day 3, day 7, day 10, and day 14 with the cages, and the

average daily weight gain (ADG) and average daily feed intake (ADFI) were calculated per

group. Feed consumption (FC) was also determined on day 3, day 7, day 10, and day 14 with

the cages, and feed consumption per chick (g/chick) was calculated by dividing the total FC of

each cage by the actual number of chicks in that cage. The feed conversion ratio (FCR) was

determined as the FC per body weight gain (g/g) per cage per time. Mortality (no breathing,

no heartbeat) was recorded on a daily basis.

Blood parameters

Before euthanasia, blood samples were obtained through wing veins using heparinized syrin-

ges. All groups were analyzed for red blood cell (RBC) count, hemoglobin (Hb) level and



hematocrit (Hct) values. These parameters were determined using an automatic blood ana-

lyzer (XFA6000, Pulang Company, Nanjing, China) that was standardized for the analysis of

chicken blood parameters.

Morphology and histology of the tibial growth plates

Two birds per treatment cage (n = 8/treatment) were randomly selected on day 3, day 7, day

10, and day 14 of the experiment. The stripping of the tibial longitudinal muscles and prepara-

tion of sagittal sections of the proximal tibial growth plates were performed to analyze the mor-

phology as previously described by Rath et al. [30]. The collected tibial bone samples were

fixed in 4% paraformaldehyde at 4˚C in PBS and decalcified in 10% EDTA. After the samples

were dehydrated in ethanol and cleared in xylene, all the samples were embedded in paraffin,

and histological sections of 4~5 μm thickness were prepared and stained with hematoxylin

and eosin for microscopic examination as previously described [19,31].

Statistical analysis

Statistical analyses of the data were performed using SPSS Statistics Version 17.0 software for

windows (SPSS Inc., Chicago, IL). Comparisons between two groups were performed using

one-way ANOVA followed by Duncan’s test. For mortality, a χ2 analysis was performed for

each group. Differences were considered statistically significant at p<0.05, and the values were

presented as the means±SD or SEM.

Results

Overall performance of the chickens

There was no significant difference in the body weight (BW) of AACs and TBCs per treatment

group from day 1 to day 10 of the experiment. However, the BW of AACs and TBCs raised under

hypoxic conditions were significantly lower on day 14 (p = 0.04 and p = 0.015, respectively)

Fig 1. Effects of high altitude hypoxia on the birth weight and body weight of AACs and TBCs. (A) Effect of high altitude hypoxia on the birth weight of

AACs and TBCs (n = 12). (B) Effect of high altitude hypoxia on the body weight of AACs and TBCs (n = 8). The data are expressed as the mean±SD.

*p<0.05, normoxia group vs. hypoxia group. BW, body weight.

doi:10.1371/journal.pone.0173698.g001



compared to the normoxia group. Furthermore, the effects of hypoxia on the broilers were pro-

gressively severe (Fig 1B).

As shown in Fig 2, there was no significant difference in the average daily feed intake

(ADFI) of the AACs compared to the normoxia group during the 14 days of experiment. Simi-

larly, there were no significant changes in the average daily weight gain (ADG) and feed con-

version ratio (FCR) of the AACs, except on day 14 (p = 0.002 and p = 0.003, respectively)

between the normoxia group and hypoxia group. However, the ADFI of the TBCs was signifi-

cantly decreased (p = 0.002) during the last four days (day 10~14) of the experiment. In con-

trast, ADG was significantly decreased in the TBCs of the normoxia group and hypoxia group

(p = 0.022 and p = 0.004, respectively). Conversely, FCR was significantly increased in the TBC

normoxia group and hypoxia group on day 10 and day 14 (p = 0.005 and p = 0.014,

respectively).

Chicken mortality rate

The rate of mortality in the AACs and TBCs was 7.5% (9/120) and 5.83% (7/120), respectively,

throughout the experiment (1~14 days). The mortality per treatment group (normoxia group

and hypoxia group) is illustrated in Fig 3. Although no significant difference was observed in

each treatment group of the AACs and TBCs (p = 0.30, OR = 2.11; p = 0.70, OR = 1.36, respec-

tively), the rate of mortality was higher for AACs than TBCs under the same conditions.

Blood parameters

In this study, there was no significant difference between the normoxia group and hypoxia

group. However, a rising tendency in the total RBC count, Hb level and Hct values (except Hb

levels of AACs) was observed among all the AAC and TBC groups from day 10. The Hb level

of AACs in the hypoxia group was significantly different on day 7 and day 14 (p = 0.024 and

Fig 2. Effect of high altitude hypoxia on the overall performance of AACs and TBCs (n = 4 cages). The value for the red line as the reference in this

figure is 2. The data are expressed as the mean±SD. *p<0.05, normoxia group vs. hypoxia group.




p = 0.033, respectively) in comparison to that of the normoxia group. In contrast, hypoxia had

a more apparent impact on the blood parameters (RBC and Hb) of AACs compared to those

of TBCs (Fig 4).

Morphological changes in the tibial growth plates

To examine the development of proximal tibial growth plates, eight chickens were randomly

selected and sacrificed from each group (normoxia group and hypoxia group) on day 3, day 7,

day 10, and day 14. As shown in Fig 5, the widths of the proximal tibial growth plates of AACs

were markedly enlarged on day 10 and day 14 in the hypoxia group. However, the widths of

the tibial growth plates of TBCs were not enlarged compared to the normoxia group. In addi-

tion, the tibial growth of AACs was much faster compared to TBCs.

Histological examination of the tibial growth plates

Histological analysis of the proximal tibia of AACs showed a significant increase in the density

of metaphyseal blood vessels on day 14 in the hypoxia group compared to the normoxia

group. However, no obvious changes in TBCs were observed (Fig 6).

Discussion

TBCs are an aboriginal breed with a history of thousands of years of living in high altitude

areas and are characterized by their small size and low birth weight, which may be attributed

to their better adaptation to altitudes of more than 4,000 m (Fig 1A). The findings of the pres-

ent study indicated the effect of high altitude hypoxia on suppressing the BW of AACs and

TBCs in comparison with the normoxia group. These observations confirmed the findings of

Gao et al. [32], which indicated reduced body weight gain during hypoxia. This reduced body

weight gain might be due to a reduction in the nutritional energy intake or intestinal energy

Fig 3. Effect of high altitude hypoxia on the mortality rate of AACs and TBCs. χ2 analysis was performed

on the number of chickens who died throughout the experiment.




uptake as a result of impaired intestinal function and increased energy expenditure [3]. Hyp-

oxia directly leads to systemic hypoxemia and an imbalance between the animal’s demand for

O2 and the insufficient O2 available, resulting in a decrease in BW at high altitudes. Semenza

[33] reported that an inadequate supply of O2 affects both physiological performance and

growth capacity. Altogether, our findings also demonstrated the various effects of high altitude

hypoxia on the overall performance of TBCs and especially AACs.

The final BW values of AACs and TBCs in the hypoxia group at 2,986 m above sea level on

day 14 were 163.75 g and 55.54 g, respectively, similar to the findings of Li et al. [3], where the

BW of AACs raised at high altitude was approximately 172.6 g on day 14. Meanwhile, previous

reports on the BW of TBCs had lower numbers, but the ADFI remained the same as the AACs.

Similarly, Westerterp et al. [34] suggested that energy intake is the dominant determinant of

body weight loss for humans under hypoxic conditions at high altitudes. Similar observations

were made by De Grauw et al. [35] and Camm et al. [36], who demonstrated that exposure to

hypoxia can lead to a significant decrease in food intake. The reduction in food intake may not

solely account for the decrease in BW, as Li et al. [3] indicated that the villi height and crypt

depth of AACs reared at high altitude were also reduced compared to those reared at low alti-

tudes. Altogether, these findings suggest that the absorption of nutrients at high altitudes

could be compromised; thus, hypoxia affects not only ADFI but also ADG. In addition, a sur-

prising and inspiring finding from this study was the value of FCR, which was lower than 2

between days 3~10 and markedly increased on day 14 under hypoxia conditions, suggesting

the induction of feed conversion to body mass. This observation is likely in agreement with

the report of Li et al. [3], who found that the FCR of broilers reared at high altitudes was 2.21

Fig 4. Effect of high altitude hypoxia on the blood parameters of AACs and TBCs (n = 4). The data are expressed as the mean±SEM. *p<0.05,

normoxia group vs. hypoxia group. RBCs, red blood cells; Hb, hemoglobin; Hct, hematocrit.




Fig 5. Effect of high altitude hypoxia on the morphology of the growth plates in AACs and TBCs. The enlarged growth plates in the hypoxia

group were compared with normoxia group. AC, articular cartilage; GP, growth plate.




on day 14. Nevertheless, the FCR was also less in the TBCs. In this study, we noticed that the

FCR of TBCs was higher than that of AACs during all four readings and that the value of FCR

was greater than 2, even up to 3.5, suggesting the prolong growth period of TBCs and low eco-

nomic benefits caused by the hypoxic environment.

The present experimental study on the production performance of broilers showed that

hypoxia not only affects the birth weight, BW, ADFI, ADG, and FCR but also slightly affects

the survival ratio of both AACs and TBCs. Similar observations were made by Visschedijk

[37], who studied lowland chickens raised at high altitudes where inadequate O2 exchange

resulted in hypoxic syndrome. In general, proper ventilation is an important strategy to avoid

high death rates in lowland chickens raised at high altitudes [38]. Noticeably, hypoxia affects

the survival rates of both embryos and lowland chickens at high altitudes. The results of the

present study did not indicate any significant difference in the mortality rates of AACs and

TBCs exposed to hypoxia or normoxia during the rearing period. However, hypoxia is known

as a major risk factor for the death of broilers, especially AACs.

Fig 6. Histological examinations of the growth plates in both AACs and TBCs at high altitude. Obvious increase in the density of the metaphyseal

blood vessels on day 14 in the hypoxia group compared to the normoxia group. The arrows indicate blood vessels. BV, blood vessels. Scale

bar = 500 μm.




Blood can be affected by hypoxia acts as transports oxygen to the organs of the body, and its

parameters are very critical in evaluating animal physiology under hypoxic conditions [39–

41]. In particular, Hb acts as a hypoxic sensor, along with the RBCs, to perform the fundamen-

tal physiological process of O2 delivery to the hypoxic tissues [39,42]. Another study by Liu

et al. [43] showed that the Hb concentration is the most important factor responsible for

ensuring oxygen concentration in the blood under hypoxic conditions. In the present study,

we found that the hypoxic environment significantly increased the Hb levels on day 14 com-

pared to the normoxia group in AACs, and similar changes in Hb have also been observed in

avian embryos [44]. Furthermore, an increasing tendency in the RBC number, Hb level and

Hct values was observed at days 10~14 in both the AAC and TBC groups during the entire

experiment. These effects could be due to an increase in the oxygen demand of the birds for

respiration and normal physiological processes, and the increased RBC number, Hb concen-

tration, and Hct volume can be attributed to the oxygen compensatory effects. However, hyp-

oxia had a more pronounced impact on the blood parameters of AACs compared to TBCs,

suggesting that the TBCs have good adaptability to hypoxic conditions. In contrast, hypoxia

influenced the bone marrow of the broiler chickens to increase the RBC number and Hb con-

centration and to increase the blood vessel number in the face of imminent hypoxia. There-

fore, further studies are required to confirm the effects of hypoxia on the growth and

development of tibial growth plates in relation to the numbers of blood vessels [45].

The most recommended methods for the assessment of tibial growth plate development

and pathology are TUNEL assays and hematoxylin and eosin staining [19,31,46,47]. Morpho-

logical examination of the tibial anatomy includes envisioning the width of the tibial growth

plate, which is considered an alternative method for the assessment of tibia development

[19,30]. Additionally, focusing on the production performance of the birds, all parameters,

including mortality rates, of both the AACs and TBCs at high altitudes were found to be closely

related to hypoxia. These findings suggest that hypoxia has a negative effect on the growth of

broiler chickens at high altitudes. However, the hypoxia group, especially the AACs that were

constantly kept under hypoxic conditions for 14 days, showed a surprising increase in the tibial

growth plate size (morphologically). At the same time, the histology of the tibial growth plates

on day 14 showed higher blood vessel densities. Previous studies have indicated that the distur-

bance of blood vessels on the growth plate decreases bone mineralization and hypertrophic

chondrocyte replacement [48,49]. Moreover, Lee et al.[50] indicated that the increased param-

eters of bone formations were closely related to high blood vessel number and density. Similar

results were reported by Zhao et al. [51], who highlighted the role of hypoxia in inducing chon-

drogenesis and angiogenesis, as well as its role in the bone repair process. Altogether, these

findings suggest that bone formation is largely dependent on vascularization. Thus, hypoxia-

induced angiogenesis for the formation of tibial growth plates is of great importance. However,

in this study we found that the width of tibial growth plates and the density of the metaphyseal

blood vessels in the proximal tibia of TBCs did not show any apparent change, unlike AACs,

which may be attributable to the adaptability of the TBCs to long-term hypoxic conditions and

a possible reason for the nonexistence of TD in these birds.

Unlike human beings, hypoxia-induced weight loss in chickens [34,52,53] is beneficial in

terms of their fitness. However, the external hypoxic environment may also prevent the occur-

rence of TD. Nevertheless, to pursue the largest economic benefits in large-scale poultry breed-

ing, it is important that the birds gain more BW; therefore, possible drug applications are

required to obtain hypoxic conditions [51]. Moreover, studies by Hsieh et al. [54] reported

that such drugs can prolong the activity of HIF-1α in the bloodstream and lead to an increase

in the endogenous production of erythropoietin, which may enhance chondrogenesis and vas-

cular formation.



In summary, our results showed the extensive effects of hypoxia at high altitudes on the

overall performance of poultry and on the development of the tibial growth plates in particu-

lar. We proposed that hypoxia not only has a negative effect of the growth performance of

broilers but also plays an important role in the enlargement of tibial growth plate sizes and in

the increase in metaphyseal blood vessel density to the proximal tibia. Altogether, these find-

ings may provide new insights for the therapeutic manipulation of hypoxia in poultry TD and

have important implications for the pathophysiology of tibial growth plates under hypoxic

conditions. However, the expression of related genes responsible for the increase in the width

of the growth plates and for the corresponding vascular density is unclear. Further studies on

the possible functional effects of growth plate-related genes, including HIF-1α, VEGF, and its

receptors (such as VEGFR1 and VEGFR2), are required.

Supporting information

S1 Table. Nutrient composition of the broiler diet.

(DOC)

Acknowledgments

The study was supported by the National Natural Science Foundation of China (No.

C180802).

Author Contributions

Conceptualization: JKL SCH.

Data curation: SCH LHZ GQ WYY MW JKL.

Formal analysis: SCH LHZ YFL.

Funding acquisition: JKL.

Investigation: SCH LHZ MKI KM HZ WYY MW.

Methodology: SCH.

Project administration: JKL.

Resources: SCH LHZ HZ GQ.

Software: SCH.

Supervision: JKL SCH.

Writing – original draft: SCH MUR.

Writing – review & editing: SCH MUR MKI FN JKL.

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high altitude hypoxia as a factor that promotes tibial growth ......citation: huang s, zhang l,...

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